The primary structure of a 38 kDa protein isolated from membrane preparations of African trypanosomes (Trypanosoma brucei rhodesiense) was determined by protein and DNA sequencing. Searching of the protein database with the trypanosome translated amino acid sequence identified glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) from various prokaryotic and eukaryotic organisms as the optimal scoring protein. Surprisingly, the eukaryotic trypanosome enzyme showed the highest degree of sequence identity with the corresponding enzyme from the prokaryote Escherichia coli. Using recombinant DNA techniques, the trypanosome molecule was expressed in Escherichia coli and found to be enzymatically active, thus confirming the identity of the molecule as an NAD+-dependent glycerol 3-phosphate dehydrogenase. A monoclonal antibody specific for the 38 kDa protein was used to localize the enzyme to glycosomes. The enzyme has a pi of 9.0, a net charge of +9 at physiological pH and contains the peroxisome-like targeting tripeptide SKM at its C-terminus, all characteristic of glycosomal enzymes. Amino acids predicted to be involved in the NAD+-dependent glycerol 3-phosphate dehydrogenase active site have diverged from those of the mammalian enzyme. Kinetic analyses of the trypanosome GPD and GPD from rabbit muscle showed that the Km values of the two enzymes are different The data suggests that the trypanosome protein may be a candidate target for rational drug design. Northern and Southern blot analyses showed that the trypanosome NAD+-dependent glycerol 3- phosphate dehydrogenase was translated from a single transcript and that only two gene copies exist thus making this molecule an attractive target for knockout mutagenesis. A second molecule, an abundant 11 kDa membrane protein, was also purified from African trypanosomes. This protein cross-reacted with monoclonal antibodies originally generated against the lipophosphoglycan-associated protein of Leishmania donovani. Immunoblot analysis showed that the 11 kDa molecule was present in a variety of species of kinetoplastids. It was found in several species and subspecies of African trypanosomes and was present in low amounts in bloodstream forms and in larger amounts in procyclic, epimastigote and metacyclic life cycle stages. The molecule was present in procyclic trypanosome membranes at approximately [special characters omitted] molecules per cell. Its wide distribution in kinetoplastids and its membrane disposition suggested a name for this class of molecules (kinetoplastid membrane protein-11) and for the molecule characterized in this thesis (trypanosome kinetoplastid membrane protein-11). The kinetoplastid membrane protein-11 molecule was purified from Trypanosoma brucei rhodesiense by organic solvent extraction and octyl-Sepharose chromatography and a 14 amino acid internal peptide sequence was obtained by gas phase microsequencing. This sequence matched a translated Leishmania donovani kinetoplastid membrane protein-11 sequence, thus suggesting the use of the Leishmania sequence as a probe to select for the Trypanosoma gene. Screening of a trypanosome cosmid library with the Leishmania probe, in combination with a series of polymerase chain reaction amplifications from both genomic DNA and cDNA, allowed the determination of the entire DNA sequence and corresponding translated amino acid sequence of the trypanosome kinetoplastid membrane protein-11. The 92 amino acid sequence showed 18 percent sequence divergence from the corresponding molecule of the related kinetoplastid Leishmania donovani donovani^ including one key amino acid at position 45 which may be of functional relevance. The secondary structure of the trypanosome molecule was predicted to form two amphipathic helices connected by a random-coil segment, and suggests that it would interact with lipid bilayers in the parasite cell membrane. Northern and Southern blot analyses using the T.b. rhodesiense ViTat 1.1 clone showed that the trypanosome molecule was translated from a single transcript and that there was only a single gene copy, thus making this molecule an attractive target for knockout mutagenesis. / Graduate

No description available.

A dissertation submitted to the faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science. Johannesburg, 2014. / Human African Trypanosomiasis (HAT) is transmitted by Glossina species and remains a serious health problem in Africa. Many aspects of control of the disease have been implemented throughout the years but vector control of tsetse flies has proven to be the most efficient long-term solution. Vector control interventions have been implemented for many tsetse species but relatively little is known about the behaviour of the riverine species, Glossina fuscipes fuscipes. Increased knowledge of this species would improve vector control interventions. This study aimed at: i) understanding the behaviour of tsetse flies around visual devices and odour baits; ii) understanding the behaviour of the flies with regard to human activities; iii) understanding the interaction between the nutritional status of tsetse flies and their attraction to various trapping devices (biconical traps and electric nets); and iv) establishing an age determination curve for field-caught flies. Results showed that visual targets were better attractants then odour-based ones and electric nets performed better than biconical traps. The sticky traps caught 10x more flies (males) than the stationary biconical traps. Sticky traps caught more young flies than the biconical traps which caught more old flies. An age curve was established for flies ranging from 1 day to 60 days old and the fluorescence-based age determination technique, using pteridine levels, has been shown to work for this species. Understanding the behaviour of tsetse flies around trapping devices should lead to improved trapping efficiency. The data gathered will be of importance in assisting with designing and running the Lake Victoria region control operations planned by PATTEC and it will have application in G. f. fuscipes endemic regions in other parts of Africa.

Thesis (M.S.)--Villanova University, 2008. / Chemistry Dept. Includes bibliographical references.

Thesis (M.S)--Villanova University, 2010. / Chemistry Dept. Includes bibliographical references.

Procyclic culture forms of Trypanosoma brucei species and antibodies to these parasites were used in developing antibody-detection and antigen-detection assays for diagnosis of African human sleeping sickness. An agglutination assay using live procyclic trypanosomes--the Procyclic Agglutination Trypanosomiasis Test (PATT) was developed for detecting anti-trypanosome antibodies in the sera of trypanosome-infected vervet monkeys and humans. Antibodies to procyclic surface antigens were detected by the PATT in sera of vervet monkeys as early as 7 days post-infection with T. b. rhodesiense. Positive agglutination titres were obtained with sera from monkeys with active, untreated infections and with sera taken soon after successful drug cure. Similar positive agglutination results were also observed using the PATT with sera from T. b. gambiense-infected patients from Cote d'Ivoire and Sudan and with documented sera from T. b. rhodesiense-infected patients from Kenya. No agglutination reactions were observed with preinfection sera from vervet monkeys, with sera from uninfected Canadians or with sera from Americans working in endemic areas. Together these results confirm the diagnostic value of using procyclic trypanosomes to detect anti-trypanosome antibodies in human African sleeping sickness. A double antibody sandwich ELISA using monoclonal antibodies and polyclonal rabbit antibodies to the surface membrane antigens of procyclic trypanosomes was developed. This assay detected circulating trypanosomal antigens in the sera of trypanosome-infected mice and in the sera from parasite-infected patients. However, limited success was obtained with this sandwich ELISA when tested on a larger repertoire of sera from infected humans. Rabbit antibodies made against whole lysates of T. b. rhodesiense procyclics were then employed in an antigen-trapping sandwich ELISA. The results demonstrated the effectiveness of this sandwich ELISA in revealing the infection status of vervet monkeys or humans infected with either T. b. rhodesiense or T. h. gambiense. Trypanosomal antigens were detected in the sera of parasitologically confirmed monkeys and patients but not in preinfection sera nor in control sera from uninfected North Americans. The PATT and the sandwich ELISA exhibited higher sensitivities than the currently employed diagnostic assay for human sleeping sickness, the Card Agglutination Trypanosomiasis Test (CATT), when tested with sera of parasitologically-confirmed humans. The sandwich ELISA was superior to the antibody-detecting PATT and CATT in monitoring trypanocidal drug-treated patients. The overall sensitivity of the PATT and sandwich ELISA was 94.3% and 97.4% and the specificity was 84.5% and 95.5%, respectively. These results thus confirm the diagnostic value of these tests for the diagnosis of human African sleeping sickness. Identification of diagnostically useful antigens was attempted in order to facilitate the adaptation of these diagnostic assays to a simpler format for field application. Pooled sera obtained from trypanosome-infected patients was used as a probe to detect trypanosome antigens separated by high performance liquid chromatography, immunoaffinity and immunoblotting techniques. Most of the antigens were detected in the higher molecular weight range (>62 Kd). Immunization of mice with the target antigens yielded six trypanosome-specific monoclonal antibodies. In a double antibody sandwich ELISA, these antibodies were successful in trapping circulating parasite antigens in sera from trypanosome-infected mice as early as 3 days post-infection. Some of these antigens have been partially biochemically characterized. Trypanosomal antigens were also detected by these antibodies in the urine of infected mice. The antigen-capture sandwich ELISA using either the selected monoclonal antibodies or the rabbit anti-procyclic whole lysate antibodies gave similar results with sera from trypanosome-infected mice, human sleeping sickness patients and uninfected humans from North America and Kenya. The results showed that these MAbs and their antigens were useful in the diagnosis of African human sleeping sickness. / Graduate

Drug resistance together with difficulties involved in the development of new trypanocides are a major problem in the present control of African trypanosomiasis. DNA based diagnostics for drug resistance would overcome problems in the identification of drug-resistant populations and contribute to effective control measures. However, this requires a detailed knowledge of the mode of action and the mechanisms by which trypanosomes can overcome the toxic effects of trypanocides. In this study, a search for molecular differences between a multidrug-resistant isolate of Trypanosoma brucei brucei, CP 547, and a reference drug-sensitive population, ILTat 1.4, led to the identification of a 6.6 kbp extrachromosomal element in the multidrug-resistant population. In light of the involvement of extrachromosomal elements in drug resistance in Leishmana spp. and cancer cells, the identification of the 6.6 kbp element warranted its characterisation. Several different approaches sere attempted before a sequence which hybridised to the 6.6 kbp element its eventually isolated. This sequence is represented by a 108 bp repeat sequence which forms long arrays of tandem repeats. Since N/a III is the sole restriction enzyme that cuts within the repeat, it has been referred to as an N/a III repeal The repeat is flanked by a 5 bp spacer sequence. However, a unique 5 bp direct repeat flanking two complete, and one partial copy of the N/a III repeat may signify the transposition of these sequences. Hybridisation with the N/a III repeat revealed the presence of 'higher' hybridising elements which also appear to be predominantly composed of long tandem arrays of the N/a Ill repeal Through exploitation of the p01) merase chain reaction using arbitrary primers (AP-PCR), additional sequences were identified which are associated with some of the 6.6 kbp and 'higher' hybridising elements. The 6.6 kbp element and some of the 'higher' hybridising elements display features of circular DNA molecules. The 6.6 kbp element also displays some level of size and sequence heterogeneity within different populations of the same trypanosome isolate. The copy number of the 6.6 kbp element is also not stable and appears to be directly affected by the application of selective drug pressure, but a direct association between the presence of the element and the expression of multidrug resistance could not be determined. The N/a III repeat family represents a newly identified repetitive family specific to members of the Trypanozoon subgenus. This repeat family, representing about 5% of the parasite genome, is dispersed through all size classes of chromosomes, in addition to its presence on the extrachromosomal elements. Transcriptional studies of the N/a III repeats have revealed that their transcription is developmentally regulated, since heterogeneous transcripts ranging from greater than 10 kb to smaller than 300 bp are present in the actively dividing long slender bloodstream and insect stage procyclic forms of the parasite but not nondividing, stumpy bloodstream forms. Lastly, the N/a III repeat lacks an open reading frame and transcripts do not appear to have a spliced leader sequence at the 5' end. Furthermore, there is almost an equal representation of polyadenylatcd and non-polyadenlyated transcripts.

No description available.

This dissertation is a history of sleeping sickness research work and prevention programs during the German colonial period in East Africa, focusing on the regions around Lake Victoria and Lake Tanganyika. It examines efforts to study and prevent sleeping sickness, analyzing how both fit into the social, political, and economic dynamics of African life. It covers two phases of German colonial attention to epidemic sleeping sickness between 1902 and 1914: an initial phase of research and scientific expeditions from 1902 to 1906, then a period dominated by the introduction of disease prevention measures in affected areas from 1907 to 1914. Highlighting the local complexity and far-reaching impact of sleeping sickness, I show that sleeping sickness research and prevention emerged from the intersection of tropical medicine expertise, African mobility, and German colonial and African politics. Sleeping sickness, and subsequent efforts toward its treatment and prevention, redefined the boundaries of political power and social influence within African communities during a crucial period of change in the region. By creating new arenas of engagement between African communities and European scientists, specifically in newly-built sleeping sickness camps and among the African medical auxiliaries employed in them, sleeping sickness work created economic relationships, reshaped social and political hierarchies, and set new ground rules for African agriculture and trade. Kings, chiefs, and colonial scientists contended with African communities' demands for treatment, their resistance to examination, and their claims on the use of land and waterways. Further, inter-colonial sleeping sickness research and subsequent prevention programs played a pivotal role in the development of tropical medicine, strengthening disciplinary boundaries and defining the trajectory of future research. My work weaves together narratives of research and disease prevention from metropolitan Europe and East Africa, in contrast to strictly colonial and national histories of health and medicine that have preceded it.

Undergraduate honors paper--Mount Holyoke College, 2008. Dept of Biological Sciences. / Includes bibliographical references (leaves 57-59).